Compositions and methods for the detection of antibodies to native human leukocyte antigen

ABSTRACT

Provided herein are compositions comprising native and denatured human leukocyte antigens (HLA) and methods of making said compositions. Also provided herein are methods and kits for the detection of antibodies to native HLAs.

1. CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C.§119 of U.S. Provisional Application No. 61/338,258, the content ofwhich is hereby incorporated by reference in its entirety.

2. FIELD OF THE INVENTION

The compositions and methods provided herein generally relate tocompositions that are useful, for example, in the detection ofantibodies to native human leukocyte antigen (HLA), and methods of theirpreparation.

3. BACKGROUND

Human leukocyte antigens (HLAs) can bind and display antigens on thesurface of human cells to effector T cells. The two major classes ofHLAs, class I and class II HLAs, present both foreign and nativeantigens. Class I HLAs can bind and present peptide antigens producedintracellularly, including viral and tumor specific proteins, to CD8+effector T cells (e.g., cytotoxic T cells (CTLs)). In response toforeign antigens presented by class I HLA bearing cells, CD8+ effector Tcells can destroy the cells presenting the foreign antigen. Class IIHLAs can bind and present peptide antigens that originateextracellularly to CD4+ T cells (e.g., helper T cells). In response toforeign antigens presented by class II HLA bearing cells, CD4+ effectorT cells can mount humoral immune responses. HLAs are thought to play arole in certain cancers and autoimmune disorders as well as graftrejection.

Antibodies to HLAs are usually produced by alloimmunization resultingfrom transfusions, pregnancies or transplants. They have also been foundin nonalloimmunized individuals. Morales-Buenrostro et al.,Transplantation 86: 1111-15 (2008). Antibodies to HLAs found intransplant recipients have been shown to be a cause of acute and chronicgraft rejection. Thus, determining whether a recipient carriesantibodies to HLAs of a donor can be important in determining the riskof graft rejection in a recipient.

To date, substrates linked to HLAs have been helpful in the detection ofantibodies to HLAs. Samples from recipients are contacted with asubstrate, and antibodies that bind to the substrate subsequently can bedetected using conventional techniques. Conventional substrates,however, are typically linked to both native and denatured HLAs. Thus,these substrates are limited in their ability to distinguish betweenantibodies to native and denatured HLAs.

In some instances, antibodies to native HLAs, but not to denatured HLAs,are predictive of graft failure. Cai et al., Transplantation 88(2):226-31(2009). Therefore, there is a need for compositions and methodscapable of detecting antibodies to native HLA without the interferenceof antibodies to denatured HLAs. Such compositions and methods can beused to prevent prospective donors from being excluded due to falsepositive signals, for example, when assays screening recipients forantibodies to donor HLAs detect, instead, antibodies to denatured HLAs.

4. SUMMARY

Provided herein are compositions and methods capable of, for example,the detection of antibodies to native HLAs. In a first aspect providedherein is a composition comprising native and denatured HLAs, whereinthe native HLAs are present in substantial amounts. In some embodiments,at least 90% of the HLAs are native and at most 10% of the HLAs aredenatured. In some embodiments, at least 95% of the HLAs are native andat most 5% of the HLAs are denatured. In some embodiments, at least 99%of the HLAs are native and at most 1% of the HLAs are denatured.

In some embodiments, the native and denatured HLAs are selected from thegroup consisting of class I HLAs, class II HLAs and combinationsthereof. In some embodiments, the HLAs are class I HLAs. In someembodiments, the HLAs are class II HLAs. In some embodiments, the HLAsare a combination of class I and class II HLAs.

The native and denatured HLAs can be of the same allele or two or moredifferent alleles. In some embodiments, at least 90% of the HLAs are ofthe same allele. In some embodiments, at least 95% of the HLAs are ofthe same allele. In other embodiments, at least 99% of the HLAs are ofthe same allele.

In some embodiments, the native and denatured HLAs are linked to a solidsubstrate. The HLAs can be linked to the solid substrate by anytechnique known to those of skill in the art. In some embodiments, theHLAs are directly linked to the solid substrate. In other embodiments,the HLAs are indirectly linked to the solid substrate.

The solid substrate can be made of any suitable material known to thoseof skill in the art. In some embodiments, the solid substrate comprisesa material selected from the group consisting of silica, gold, latex,polystyrene, polyethylene, polysulfone, hydrogel, polyvinyl chloride,glass, and combinations thereof.

Further, the form of solid substrate can be any form deemed suitable bythose of skill in the art. In some embodiments, the solid substrate isselected from the group consisting of a plurality of beads, a pluralityof microbeads, a plurality of microparticles, a plurality ofmicrospheres, a well, a membrane, a polymer, a filter, a microarray andcombinations thereof. In some embodiments, the solid substrate is aplurality of microbeads.

In some embodiments, the solid substrate comprises a detectable label.In some embodiments, the detectable label comprises a fluorescent dye, aradioactive label, a magnetic label or a bar code. In certainembodiments, the detectable label is a fluorescent dye.

In another aspect provided herein are panels comprising a plurality ofsolid substrates, wherein each solid substrate of the plurality islinked to HLAs, wherein at least 90% of the HLAs linked are native andat most 10% of the HLAs are denatured, w herein at least 90% of the HLAslinked to a particular solid substrate of the plurality are of the sameallele, and wherein each solid substrate of the plurality is linked to adifferent HLA with respect to the other solid substrates of theplurality. In some embodiments, the HLAs comprise HLAs selected from thegroup consisting of class I HLAs, class II HLAs and combinationsthereof. In some embodiments, the plurality comprises 4 solidsubstrates, 8 solid substrates, 16 solid substrates, or 32 solidsubstrates.

In another aspect provided herein are methods for making a compositioncomprising at least 90% native HLAs and at most 10% denatured HLAs. Themethods comprise: a) contacting a first composition comprising nativeand denatured HLAs with a serine protease, lipase, esterase, or amidaseunder conditions wherein the serine protease, lipase, esterase, oramidase cleaves the denatured HLAs; and b) neutralizing the serineprotease, lipase, esterase or amidase to yield a resulting compositioncomprising at least 90% native HLAs and at most 10% denatured HLAs. Incertain embodiments, the HLAs are suspended in solution. In certainembodiments, the HLAs are linked to one or more solid substrates. Inparticular embodiments, the HLAs are linked to a plurality of microbeadsor microparticles.

In some embodiments of the methods, the HLAs are contacted with a serineprotease. In particular embodiments, the serine protease is trypsin. Insome embodiments, the HLAs are contacted with a lipase. In particularembodiments, the lipase is a phospholipase. In some embodiments, theHLAs are contacted with an esterase. In particular embodiments, theesterase is acetylcholine esterase. In other embodiments, the esteraseis a thioesterase. In some embodiments, the HLAs are contacted with anamidase.

In another aspect provided herein is a method of screening forantibodies to native HLAs. This method comprises the steps of: a)contacting a sample with a composition comprising HLAs linked to a solidsubstrate, wherein at least 90% of the HLAs are native and at most 10%of the HLAs are denatured; and b) detecting binding of an antibody tothe composition, wherein binding of an antibody to the composition isindicative of antibodies to native HLAs.

In some embodiments, the sample is a biological sample. In someembodiments, the sample is taken from a human subject. In someembodiments, the sample is a blood sample taken from a human subject.

Detection of binding of antibody to the composition can be performed byany technique known to those of skill the art. In some embodiments, thedetection of antibody binding is performed using flow cytometry. In someembodiments, detection of antibody binding is performed using asecondary antibody. In particular embodiments, the secondary antibodycomprises a label selected from the group consisting of a radioactivelabel, a fluorescent label, an enzymatic label, an avidin label, abiotin label and combinations thereof.

In another aspect provided herein are kits for the detection ofantibodies to native HLA. These kits comprise: a) a compositioncomprising HLAs linked to a solid substrate, wherein at least 90% of theHLAs are native and at most 10% of the HLAs are denatured, and b) areagent for detecting the binding of antibodies to the composition. Insome embodiments, the reagent comprises a secondary antibody. In certainembodiments, the secondary antibody comprises a detectable labelselected from the group consisting of a radioactive label, a fluorescentlabel, an enzymatic label, an avidin label, a biotin label andcombinations thereof.

The compositions and methods provided herein advantageously allow forthe detection of antibodies to native HLAs without the undesireddetection of antibodies to denatured HLAs. Detection of antibodies thatare specific for native HLAs is useful in certain instances, forexample, when the presence of antibodies to native, but not denatured,HLAs is predictive of graft failure. Under these circumstances, thecompositions and methods provided herein can help prevent prospectivedonors from being excluded due to false positive signals caused by thedetection of antibodies to denatured HLAs in recipients.

5. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C provide graphs depicting the reactivity of an antibodyspecific for native class I HLA (W6/32) and denatured class I HLA (HC10)with LABScreen Single Antigen Beads (LSAB) for A-locus (FIG. 1A),B-locus (FIG. 1B) and C-locus (FIG. 1C) class I HLAs. They demonstratethe reactivity of both W6/32 and HC10 with LSAB.

FIGS. 2A-2C provide graphs depicting the reactivity of W6/32 and HC10with compositions (“native beads”) for A-locus (FIG. 2A). B-locus (FIG.2B) and C-locus (FIG. 2C) class I HLAs. The substrates used in thesestudies are microbeads. These graphs demonstrate the low reactivity ofHC10 with compositions compared to LSAB.

FIGS. 3A-3C provide graphs depicting the reactivity of threenon-alloimmunized male sera with LSAB and compositions (“native beads”)for A-locus (FIG. 3A), B-locus (FIG. 3B), and C-locus (FIG. 3C) H LAalleles. The substrates used in these studies are microbeads. Thesegraphs demonstrate the improved accuracy of compositions compared toLSAB.

6. DETAILED DESCRIPTION OF THE EMBODIMENTS 6.1 Definitions

As used herein, the terms “native,” “native human leukocyte antigen.”and “native HLA” refer to an HLA or fragment thereof that maintains thestructural and antigenic property of the extracellular portion of an HLAin its native state.

As used herein, the terms “native class I human leukocyte antigen,” and“native class I HLA” refer to a class I human leukocyte antigen orfragment thereof that maintains the structural and antigenic integrityof the extracellular portion of a class I HLA in its native state,including comprising a β2 microglobulin domain noncovalently bound to aheavy chain or fragment thereof. In some embodiments, native class IHLAs are capable of binding to W6/32 and BIH antibodies (One Lambda,Inc.).

As used herein, the terms “native class II human leukocyte antigen,” and“native class II HLA” refer to a class II human leukocyte antigen orfragment thereof that maintains the structural and antigenic integrityof the extracellular portion of a class II HLA in its native state,including comprising a heterodimer that comprises two glycosylatedpolypeptide chains noncovalently bound to each other. In someembodiments, native class II HLAs are capable of binding to HB-145(directed to HLA-DP, HLA-DQ, HLA-DR) and HB-180 (directed to HLA-DQ andHLA DR) antibodies (American Type Cultural Collection (ATCC)).

As used herein, the terms “denatured,” “denatured human leukocyteantigen,” and “denatured HLA” refer to an HLA comprising anextracellular domain that is not in a native confirmation.

As used herein, the terms “denatured class I human leukocyte antigen,”and “denatured class I HLA” refer to class I human leukocyte antigen orfragment thereof that lacks a β2 microglobulin domain. In someembodiments, denatured class I HLAs are capable of binding to HC10 (OneLambda, Inc,) and HB296 antibodies (ATCC).

As used herein, the terms “denatured class II human leukocyte antigen”and “denatured class II HLA” refer to a class II human leukocyte antigenor fragment thereof that is in a monomeric confirmation. In someembodiments, denatured class II HLAs are capable of binding HB-298(directed to HLA-DR α chain) antibody (ATCC).

As used herein, the term “solid substrate” refers to any solid substratethat is capable of binding HLAs and is compatible with the methodsprovided herein. Examples of solid substrates include a plurality ofbeads, a plurality of microbeads, a plurality of microparticles, aplurality of microspheres, a well, a membrane, a polymer, a filter, amicroarray and combinations thereof.

As used herein, the term “same HLA allele” refers to two or more HLAmolecules or fragments thereof that share similar structure andantigenic properties and are derived from the same HLA gene loci andalleles.

As used herein, the term “different HLA allele” refers to HLA moleculesor fragments thereof that possess different structure and antigenicproperties and are derived from different HLA gene loci and alleles.

6.2 Compositions for the Detection of Antibodies to Native HLA

Provided herein is a composition comprising at least 90% native HLAs andat most 10% denatured HLAs. In certain embodiments, the composition canbe used for the detection of antibodies to native HLAs. Detection ofantibodies to native HLAs can be useful, for example, in determining thelikelihood of a graft rejection. In certain embodiments, the compositioncan also be useful in the development of HLA vaccines and effector Tcell binding assays.

The composition can be made by any technique apparent to one of skill inthe art, including the methods described herein. Each element of thecomposition is discussed in further detail below.

6.2.1. Native HLA

In some embodiments, the composition provided herein comprises at least90% native HLAs and at most 10% denatured HLAs. In some embodiments, theHLAs are linked to a solid support. In other embodiments, the HLAs arein solution.

Native class I HLAs are 57 kDa glycoproteins that are present on mostnucleated human cells. Native class I HLAs typically comprise a 45 kDapolypeptide heavy chain bound to a light chain that comprises a 12 kDaβ2 microglobulin domain. In certain embodiments, the heavy chain isnoncovalently bound to the light chain. The heavy chain typicallycomprises three α subunits, a transmembrane domain, and a cytoplasmictail. The α1 and α2 subunits form a binding groove for peptide ligandbinding. A denatured class I HLA, in contrast, lacks a β2 microglobulindomain.

The class I HLA heavy chain is encoded by one of three major genes(HLA-A, HLA-B and HLA-C) or one of three minor genes (HLA-E, HLA-F andHLA-G). Allelic variation within each of these gene loci helpscontribute to the polymorphism exhibited by class I HLA. A particularclass I HLA can be categorized by the gene locus and the particularallele from which the class I HLA is expressed. Exemplary class I HLAalleles are listed in Table 1 (HLA-A alleles), Table 2 (HLA-B alleles)and Table 3 (HLA-C alleles).

Native class II HLAs are polymorphic 61 kDa heterodimeric proteins thatare present on the surface of specialized antigen presenting cells(e.g., B lymphocytes, dendritic cells, and macrophages). Class II HLAsare divided into three subclasses: HLA-DP, HLA-DQ and HLA-DR. A nativeclass II HLA typically comprises an α chain and β chain bound to eachother. In certain embodiments, the α chain and β chain are noncovalentlybound to each other. In contrast, denatured class II HLAs are monomericproteins.

Each chain of a class II HLA comprises an extracellular domain, atransmembrane domain and a cytoplasmic tail. There are six major classII HLA genes (HLA-DPA1, HLA-PB1, HLA-DQA1, HLA-DQB1, HLA-DRA andHLADRBI), each gene encoding either an α or β chain. Similar to theclass I HLA genes, each class II HLA gene comprises many alleles. ClassI and class II HLAs can be made using any technique known to those ofskill in the art including recombinant DNA techniques as described inPei et al., Transplantation 75(1): 43-49 (2003).

In some embodiments, the composition comprises a substantial amount ofnative HLAs. In some embodiments, at least 90% of the HLAs are nativeand at most 10% of the HLAs are denatured. In some embodiments, at least95% of the HLAs are native and at most 5% of the HLAs are denatured. Insome embodiments, at least 99% of the HLAs are native and at most 1% ofthe HLAs are denatured. In some embodiments, at least 99.5% of the HLAsare native and at most 0.5% of the HLAs are denatured. Techniques formaking these compositions are described below.

In some embodiments, the composition comprises HLAs selected from thegroup consisting of class I HLAs, class II HLAs and combinationsthereof. In some embodiments, the composition comprises class I HLAs. Insome embodiments, the composition comprises class II HLAs. In someembodiments, the composition comprises a combination of class I andclass II HLAs.

The composition can comprise full length native HLAs or fragmentsthereof. In some embodiments, the composition comprises full lengthnative class I HLAs, each class I HLA comprising a heavy chainnoncovalently bound to a β2 microglobulin chain. In some embodiments,the composition comprises full length class II HLAs, each class II HLAcomprising an α chain noncovalently bound to a β chain.

In some embodiments, each HLA comprises a modification such as andeletion, addition or amino acid substitution that does not disrupt thestructural and antigenic integrity of the native HLA extracellulardomain. In some embodiments, each HLA is a class I HLA that comprises anextracellular domain of a native class I HLA. In some embodiments, eachHLA is a class I HLA that comprises an extracellular domain and afragment of a transmembrane domain of a native class I HLA. In someembodiments, each HLA is a class I HLA that comprises an extracellulardomain and a transmembrane domain of a native class I HLA. In someembodiments, each HLA is a class I HLA that comprises an extracellulardomain, a transmembrane domain and a fragment of a cytoplasmic tail of anative class I HLA.

In some embodiments, each HLA is a class II HLA that comprises anextracellular domain of a native class II HLA. In some embodiments, eachHLA is a class II HLA that comprises an extracellular domain and afragment of a transmembrane domain of a native class II HLA. In someembodiments, each HLA is a class II HLA that comprises an extracellulardomain and a transmembrane domain of a native class II HLA. In someembodiments, each HLA is a class II HLA that comprises an extracellulardomain, a transmembrane domain and a fragment of a cytoplasmic tail of anative class II HLA.

The composition provided herein can comprise HLAs of the same allele orto two or more different alleles. HLAs that are of the same allele sharethe same structure and antigenic properties and are derived from thesame HLA loci and alleles.

In some embodiments, at least 90% of the HLAs are of the same allele. Insome embodiments, at least 95% of the HLAs are of the same allele. Inother embodiments, at least 99% of the HLAs are of the same allele.

In some embodiments, at least 90% of the HLAs are of the same allele andcomprise an HLA-A class I HLA. In some embodiments, at least 90% of theHLAs are of the same allele and comprise an HLA-A class I HLA that islisted in Table 1. In some embodiments, at least 90% of the HLAs are ofthe same allele and comprise an HLA-B class I HLA. In some embodiments,at least 90% of the HLAs are of the same allele and comprise an HLA-Bclass I HLA that is listed in Table 2. In some embodiments, at least 90%of the HLAs are of the same allele and comprise an HLA-C class I HLA. Insome embodiments, at least 90% of the HLAs are of the same allele andcomprise an HLA-C class I HLA that is listed in Table 3.

In some embodiments, at least 90% of the HLAs are of the same allele andcomprise an HLA-DP class II HLA. In some embodiments, at least 90% ofthe HLAs are of the same allele and comprise an HLA-DQ class II HLA. Insome embodiments, at least 90% of the HLAs are of the same allele andcomprise an HLA-DR class II HLA.

6.2.2. Solid Substrates

In certain embodiments, the HLAs provided herein are linked to a solidsubstrate. Native HLAs linked to the solid substrate allow thecomposition to bind antibodies to native HLAs. The bound antibodies tonative HLAs can be detected using any technique known to those of skillin the art.

In certain embodiments, a substantial amount of the HLAs linked to thesolid substrate are native. As used herein, a “substantial amount” canbe any amount that allows for the binding and detection of antibodies tonative HLAs without significant binding of antibodies specific fordenatured HLAs. This amount can be expressed as a percentage of thetotal number of native HLAs to total number of HLAs linked to the solidsubstrate. In some embodiments, at least 75% of the HLAs are native. Insome embodiments, at least 80% of the HLAs are native. In someembodiments, at least 85% of the HLAs are native. In some embodiments,at least 90% of the HLAs are native. In some embodiments, at least 95%of the HLAs are native. In some embodiments, at least 99% of the HLAsare native. In some embodiments, at least 99.5% of the HLAs are native.

HLAs can be linked to the solid substrate by any technique known tothose of skill in the art. Further, HLAs can be directly or indirectlylinked to the solid substrate. In some embodiments. HLAs are directlylinked to the solid substrate. In some embodiments, HLAs are directlylinked to the solid substrate by absorption, chemical coupling or bychemical linkage through a tail element added to the HLA. In certainembodiments. HLAs are directly linked to the substrate by passiveabsorption. Cantarero et al., Anal. Biochem., 105: 373-382 (1980). Insome embodiments, HLAs are indirectly linked to the solid substrate by alinking moiety. In some embodiments, the linking moiety is selected fromthe group consisting of an antibody, a lectin, a CD8 molecule, a CD4molecule, a T cell receptor and fragments thereof. In some embodiments,the linking moiety is a bifunctional cross-linker. Useful bifunctionalcross-linkers are known to those of skill in the art.

The solid substrate can be made of any material known to those of skillin the art that is able to link to HLAs. Well known materials for solidsubstrates include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, polysulfone, hydrogel, polyvinyl and magnetite. In someembodiments, the solid substrate comprises a material selected from thegroup consisting of silica, gold, latex, polystyrene, polyethylene,polysulfone, hydrogel, polyvinyl chloride, glass, and combinationsthereof.

Further, the solid substrate can have any structural configurationdeemed suitable by those of skill in the art. Solid substrates cancomprise a plurality of beads, a plurality of microbeads, a plurality ofmicroparticles, a plurality of microspheres, a well, a membrane, apolymer, a filter, a microarray and combinations thereof.

In some embodiments, the solid substrate is a plurality of microbeads.Useful microbeads are commercially available from sources such asLuminex, Inc., Invitrogen Corp. Polysciences, Inc. and BangsLaboratories, Inc. to name a few. In certain embodiments, the microbeadsare 2 to 8 μm in diameter. In certain embodiments, the microbeads are 4to 6 μm in diameter.

In some embodiments, the solid substrate is a plurality ofmicroparticles. In some embodiments, the microparticles are nanocrystalsor quantum dots.

The solid substrate can also comprise a detectable label or any otheridentifying characteristic that can allow for the identification,separation and classification of antibodies bound to the HLAs. Forexample, the substrate can be a plurality of microbeads, each labeledwith a fluorophore that allows the microbeads to be sorted using flowcytometry. Detectable labels can include fluorescent dyes, radioactivelabels, magnetic labels, bar codes and combinations thereof. In certainembodiments, the detectable label is a fluorescent dye. In certainembodiments, the detectable label is a radioactive label. In certainembodiments, the detectable label is a bar code. In certain embodiments,the substrate comprises a detectable label selected from the groupconsisting of a fluorescent dye, a radioactive label, a magnetic label,a bar code and combinations thereof.

6.3 Panels

In another aspect provided herein are panels comprising a plurality ofsolid substrates, wherein each solid substrate of the plurality islinked to HLAs, wherein at least 90% of the HLAs linked are native andat most 10% of the HLAs are denatured, w herein at least 90% of the HLAslinked to a particular solid substrate of the plurality are of the sameallele, and wherein each solid substrate of the plurality is linked to adifferent HLA with respect to the other solid substrates of theplurality. The panels can be linked to any of the HLAs provided herein.Panels advantageously allow for the detection of multiple antibodies toone or more native class I HLAs at a time.

In some embodiments, the plurality comprises at least 2 or more solidsubstrates. In some embodiments, the plurality comprises at least 4 ormore solid substrates. In some embodiments, the plurality comprises atleast 8 or more solid substrates. In some embodiments, the pluralitycomprises at least 16 or more solid substrates. In some embodiments, theplurality comprises at least 32 or more solid substrates. In someembodiments, the plurality comprises at least 64 or more solidsubstrates. In some embodiments, the plurality comprises at least 128 ormore solid substrates. In some embodiments, the plurality comprises atleast 256 or more solid substrates.

In some embodiments, each solid substrate of the plurality is linked toHLA-A class I HLAs. In some embodiments, each solid substrate of theplurality is linked to HLA-B class I HLAs. In some embodiments, eachsolid substrate of the plurality is linked to HLA-C class I HLAs. Insome embodiments, each solid substrate of the plurality is linked to anyclass I HLA selected from the class I HLAs found in Tables 1, 2, and 3and combinations thereof.

In some embodiments, each solid substrate of the plurality is linked toHLA-DP class II HLAs. In some embodiments, each solid substrate of theplurality is linked to HLA-DQ class II HLAs. In some embodiments, eachsolid substrate of the plurality is to HLA-DR class II HLAs.

In some embodiments, the panel comprises a plurality of substrateslinked to HLAs selected from the group consisting of HLA-A, HLA-B,HLA-C, HLA-DP, HLA-DQ, HLA-DR and combinations thereof.

In some embodiments, at least 90% of the HLAs linked to a particularsolid substrate are of the same allele. In some embodiments, at least95% of the HLAs linked to a particular solid substrate are of the sameallele. In some embodiments, at least 99% of the HLAs linked to aparticular solid substrate are of the same allele. In some embodiments,at least 99.5% of the HLAs linked to a particular solid substrate are ofthe same allele.

6.4 Methods for Making Compositions Useful for the Detection ofAntibodies to Native HLAs

In another aspect of the invention provided herein are methods formaking compositions comprising at least 90% native human leukocyteantigens and at most 10% denatured human leukocyte antigens. In someembodiments, the method comprises: a) contacting a first compositioncomprising native and denatured HLAs with a serine protease, lipase,esterase, or amidase under conditions wherein the serine protease,lipase, esterase or amidase cleaves the denatured HLAs; and b)neutralizing the serine protease, lipase, esterase or amidase to yield aresulting composition comprising at least 90% native human leukocyteantigens and at most 10% denatured human leukocyte antigens. The methodscan be used to make any of the compositions comprising at least 90%native HLAs and at most 10% denatured HLAs provided herein.

6.4.1. Composition of Native and Denatured HLAs

The HLAs of the first composition can comprise one or more classes ofHLAs. In some embodiments, the HLAs comprise HLAs selected from thegroup consisting of class I HLAs, class II HLAs and combinationsthereof. In some embodiments, the HLAs comprise class I HLAs. In someembodiments, the HLAs comprise class II HLAs. In some embodiments, theHLAs comprise a combination of class I and class II HLAs.

In some embodiments, the HLAs of the first composition can be insolution. In other embodiments, the HLAs are attached to a solidsubstrate.

In some embodiments, the HLAs of the first composition are suspended insolution. The HLAs can be suspended or solubilized in any solutiondeemed suitable to those of skill in the art. In certain embodiments,the solution is a buffer solution. In certain embodiments, the solutioncomprises additives that can help to prevent the nonspecific degradationof the HLAs but allow for specific cleavage of denatured HLAs. Examplesof such additives include protease inhibitors, antimicrobial agents,metal chelators and reducing agents.

In some embodiments, the HLAs are in a solution comprising phosphatebuffer solution (PBS). In some embodiments, the HLAs are in a solutioncomprising a metal chelator. In certain embodiments, the metal chelatoris ethylenediaminetetraacetic acid (EDTA). In some embodiments, the HLAsare in a solution comprising an antimicrobial agent. In certainembodiments, the antimicrobial agent is sodium azide. In certainembodiments, the antimicrobial agent is thimerosal. In certainembodiments, the HLAs are in a solution comprising a reducing agent. Incertain embodiments, the reducing agent is dithiothreitol (DTT). Incertain embodiments, the reducing agent is 2-mercapthoethanol (2-ME).

Further, in some embodiments, the solution can comprise proteaseinhibitors that help to prevent nonspecific degradation of the nativeand denatured HLAs, but allow for cleavage of the denatured HLAs. Insome embodiments, the solution comprises a protease inhibitor selectedfrom the group consisting of an acid protease inhibitor, a thiolprotease inhibitor, a metalloprotease inhibitor and combinationsthereof. Examples of such inhibitors include pepstatin A (acid proteaseinhibitor), leupeptin (thiol protease inhibitor), antipain (thiolprotease inhibitor). EDTA (metalloprotease inhibitor) and EGTA(metalloprotease inhibitor).

In some embodiments, the HLAs of the first composition are attached to asolid substrate. Solid substrates linked to native and denatured HLAscan be obtained using any technique known to those of skill in the art.Solid substrates linked to native and denatured HLAs can be obtained,for example, from a commercial source, include commercially availablemicrobeads linked to either class I HLAs, class II HLAs or combinationsthereof (e.g., FlowPRA class I and class II beads, LABScreen class I andclass II PRA beads, LABScreen Mixed beads and LABScreen Single Antigenclass I and class II beads (One Lambda, Inc.)). In other embodiments,solid substrates linked to native and denatured HLAs can be made byattaching recombinant native and denatured HLAs to a solid substrate.Recombinant native and denatured HLAs can be made and attached to solidsubstrates using any technique known to those of skill in the art,including, for example, techniques described in Pei et al.,Transplantation 75(1): 43-49 (2003).

6.4.2. Cleavage of Denatured HLAs with a Serine Protease, Lipase,Esterase or Amidase

In some embodiments, the methods comprise a step of contacting the firstcomposition of native and denatured HLAs with a serine protease, lipase,esterase, or amidase under conditions wherein the serine protease,lipase, esterase or amidase cleaves the denatured HLAs.

Any serine protease, lipase, esterase or amidase known to those of skillin the art to cleave denatured HLAs can be contacted with the substrate.Ideally, the serine protease, lipase, esterase or amidase selectivelycleaves denatured HLAs but leaves native HLAs intact. Further, theability of a particular serine protease, lipase, esterase or amidase toselectively cleave denatured HLAs can vary with the temperature andduration of time the HLAs are contacted with the particular serineprotease, lipase, esterase or amidase. One of skill in the art will beable to determine what the appropriate conditions can be for aparticular protease, lipase, esterase or amidase to cleave denaturedHLAs of a particular class.

In some embodiments of the methods, the HLAs are contacted with a serineprotease that is capable of selectively cleaving denatured HLAs whileleaving native HLAs intact. In particular embodiments, the HLAs arecontacted with a serine protease from the trypsin-like clan, whichincludes trypsin, chymotrypsin, and elastase. In particular embodiments,the serine protease is trypsin. In particular embodiments, the serineprotease is chymotrypsin. In particular embodiments, the serine proteaseis elastase. In other embodiments, the serine protease is subtilisin.

In some embodiments, the HLAs are contacted with a lipase that iscapable of selectively cleaving denatured HLAs in the first compositionwhile leaving native HLAs intact. In particular embodiments, the lipaseis a phospholipase. In particular embodiments, the phospholipase isselected from the group consisting of phospholipase A, phospholipase B,phospholipase C and phospholipase D.

In some embodiments, the HLAs are contacted with an esterase that iscapable of selectively cleaving denatured HLAs in the first compositionwhile leaving native HLA intact. In particular embodiments, the esteraseis acetylcholine esterase. In other embodiments, the esterase is athioesterase. In particular embodiments, the thioesterase is selectedfrom the group consisting of acetyl-coA hydrolase, palmitoyl-coAhydrolase, succinyl-coA hydrolase and acyl-coA hydrolase.

In some embodiments, the HLAs are contacted with an amidase that iscapable of selectively cleaving denatured HLAs while leaving native HLAsintact. In some embodiments, the amidase is peptide amidase.

6.4.3. Neutralization of the Serine Protease, Lipase, Esterase orAmidase

After cleavage of denatured HLAs by protease, lipase, esterase oramidase, the enzymatic activity of the protease, lipase, esterase oramidase is neutralized. Neutralization of a particular protease, lipase,esterase or amidase can be performed using any technique known to thoseof skill in the art. For example, neutralization can be achieved bycontacting a particular protease, lipase, esterase or amidase with areagent comprising an inhibitor of said protease, lipase, esterase oramidase.

In some embodiments, neutralization is achieved by contacting a proteasewith a reagent comprising one or more protease inhibitors. In someembodiments, the protease inhibitor is a serine protease inhibitor. Inparticular embodiments, the serine protease inhibitor is a trypsininhibitor. In particular embodiments, the trypsin inhibitor isaprotinin. In particular embodiments, the trypsin inhibitor isbenzamidine. In particular embodiments, the trypsin inhibitor isphenylmethylsufonyl fluoride (PMSF). In particular embodiments, thetrypsin inhibitor is Trypsin Neutralization Solution (Lonza, CC-5002).In particular embodiments, the serine protease inhibitor is achymotrypsin inhibitor. In particular embodiments, the chymotrypsininhibitor is chymotrypsin inhibitor 2. In particular embodiments, theserine protease inhibitor is an elastase inhibitor. In some embodiments,the elastase inhibitor is alpha 1-antitrypsin. In certain embodiments,the serine protease inhibitor is a subtilisin inhibitor. In certainembodiments, the subtilisin inhibitor is Streptomyces subtilisininhibitor.

In some embodiments, neutralization is achieved by contacting a lipasewith one or more lipase inhibitors. In certain embodiments, the lipaseinhibitor is lipistatin. In some embodiments, neutralization is achievedby contacting an esterase with one or more esterase inhibitors. In someembodiments, the esterase inhibitor is an acetylcholinesteraseinhibitor. In some embodiments, the acetylcholineserase inhibitor is anorganophosphate or a carbamate. In some embodiments, the esteraseinhibitor is a thioesterase inhibitor. In some embodiments, thethioesterase inhibitor is 5-(furan-2-ylmethylene)pyrimidine-2,4,6-trione. In some embodiments, neutralization is achievedby contacting an amidase with one or more amidase inhibitors. In someembodiments, the amidase inhibitor is a nitrile. In some embodiments,the nitrile is bis-p-nitrophenyl phosphate.

6.5 Methods for the Detection of Antibodies to Native HLAs

In another aspect provided herein are methods for the detection ofantibodies to native HLAs. Detection of antibodies to native HLAs can beuseful, for example, in the context of tissue or organ grafting, wheredetection of antibodies against donor native HLAs can be helpful indetermining the risk of graft rejection.

In certain embodiments, these methods comprise the steps of: a)contacting a sample with a composition comprising HLAs linked to a solidsubstrate, wherein at least 90% of the HLAs are native and at most 10%of the HLAs are denatured; and b) detecting binding of an antibody tothe composition, wherein binding of an antibody to the solid substrateis indicative of the presence of antibodies specific for native HLAs inthe sample. The composition can comprise any of the compositionscomprising HLAs linked to a solid substrate, wherein at least 90% of theHLAs are native and at most 10% of the HLAs are denatured providedherein.

In certain embodiments, at least 90% of the HLAs linked to the solidsubstrate are of the same allele. In certain embodiments, at least 95%of the HLAs linked to the solid substrate are of the same allele. Incertain embodiments, at least 99% of the HLAs linked to the solidsubstrate are of the same allele. In certain embodiments, at least 99.5%of the HLAs linked to the solid substrate are of the same allele.

In certain embodiments, the HLAs are selected from the group consistingof class I HLAs, class II HLAs and combinations thereof. In certainembodiments, the HLAs are class I HLAs. In certain embodiments, the HLAsare class II HLAs. In certain embodiments, the HLAs are a combination ofclass I and class II HLAs.

In certain embodiments, the composition comprises a plurality of saidsolid substrates, wherein at least 90% of the HLAs linked to aparticular solid substrate of the plurality are of the same allele andeach solid substrate of the plurality is linked to different HLA alleleswith respect to the other solid substrate of the plurality. In certainembodiments, the plurality comprises 4 or more solid substrates. Incertain embodiments, the plurality comprises 8 or more solid substrates.In certain embodiments, the plurality comprises 16 or more solidsubstrates. In certain embodiments, the plurality comprises 32 or moresolid substrates.

Any useful sample known to those of skill in the art can be used,including biological samples. Exemplary useful biological samplesinclude whole blood, blood derivatives, red blood cell concentrates,plasma, serum, fresh frozen plasma, whole blood derived plateletconcentrates, globulin, cryoprecpitate, cerebrospinal fluid, tissues andcells such as epithelial cells, such as those collected from the buccalcavity, stem cells, leukocytes, neutrophils and granulocytes.

A sample can be obtained from a human donor of a tissue, cell or organtransplantation or the intended recipient of said tissue, cell or organtransplantation. In some embodiments, the sample is obtained from ahuman donor of a kidney, liver or heart transplantation. In someembodiments, the sample is obtained from the tissue, cells or organintended for transplantation in a human recipient. In particularembodiments, the sample is obtained from a kidney, liver or heartintended from transplantation in a human recipient. In otherembodiments, the sample is obtained from a human donor or recipient of ablood transfusion. In other embodiments, the sample is obtained fromblood or blood derivatives intended for transfusion in a recipient. Incertain embodiments, the sample is obtained from a human donor orrecipient of a bone marrow transplantation.

Any system capable of detecting the binding of antibody to substrateknown to those of skill in the art can be used to detect binding of anantibody. For example, detection of binding of antibodies to native HLAscan be performed using secondary antibodies conjugated to a detectablelabel such as a radioactive label, a fluorescent label, an enzymaticlabel, an avidin label, a biotin label, or combinations thereof.Further, substrates comprising detectable labels such as those describedherein allow for multiplexed detection and classification of boundantibodies to native HLAs.

In particular embodiments, detection of antibody binding is performedusing an immunosorbent sandwich assay such as an enzyme linkedimmunosorbent assay (ELISA) assay. Immunosorbent sandwich assays can beparticular useful when the solid substrate used is a microtiter platewell. For membrane or filter solid substrates, detection of antibodybinding can be performed using immunoblotting techniques. In otherembodiments, detection of antibody binding is performed using flowcytometry.

6.6 Kits

In another aspect provided herein, are kits for the detection ofantibodies to native HLAs. In certain embodiments, the kits comprise a)a composition comprising HLAs linked to a solid substrate, wherein atleast 90% of the HLAs are native and at most 10% of the HLAs aredenatured, and b) a reagent for the detection of binding of antibody tothe composition. The composition can comprise any of the compositionscomprising HLAs linked to a solid substrate, wherein at least 90% of theHLAs are native and at most 10% of the HLAs are denatured providedherein.

In certain embodiments, the composition comprises a plurality of saidsolid substrates, wherein at least 90% of the HLAs linked to aparticular solid substrate of the plurality are of the same allele andeach solid substrate of the plurality is linked to different HLA alleleswith respect to the other solid substrate of the plurality. In certainembodiments, the plurality comprises 4 or more solid substrates. Incertain embodiments, the plurality comprises 8 or more solid substrates.In certain embodiments, the plurality comprises 16 or more solidsubstrates. In certain embodiments, the plurality comprises 32 or moresolid substrates.

Any reagent useful for detection of antibody binding known to those ofskill in the art can be used. Secondary antibodies, for example, can beuseful for the detection of antibody binding in assays such as Westernblots, immunoblots, flow cytometry, ELISAs and radioimmunoassay (RIAs).In some embodiments, the reagent comprises a secondary antibody. Incertain embodiments, the secondary antibody is an anti-human IgGantibody.

In some embodiments, the reagent comprises a secondary antibodycomprising a detectable label. In certain embodiments, the kit comprisesa detectable label selected from the group consisting of a radioactivelabel, a fluorescent label, an enzymatic label, an avidin label, abiotin label, and combinations thereof.

In some embodiments, the kits can further comprise wash buffers, controlbuffers, gels, loading buffers, molecular weight markers, platescomprising a plurality of wells and positive and negative controlsamples.

The following non-limiting examples illustrate certain embodimentsdescribed above.

7. EXAMPLES

The following examples are presented to further describe aspects of theinvention. Example 1 describes a preparation of native class I HLAmicrobeads. Example 2 describes an assay method used to identify thetypes of class I HLAs on the native class I HLA microbeads. Example 3provides analysis results to show the purity of the native class I HLAmicrobeads. Example 4 presents antibody reactivity results of the nativeclass I HLA microbeads. In these examples, commercially availableLABScreen products from One Lambda, Inc. are used and are identified bytheir catalogue numbers.

7.1 Example 1 Preparation of Microbeads Linked to Native Class I HLAS

The present example provides an exemplary preparation of microbeadslinked to native class I HLAs (“native class I HLA microbeads”).LABScreen Single Antigen Beads (LSAB), specifically, LABScreen® SingleAntigen HLA Class I—Combi (Catalogue ID: LS1A04), were obtained from OneLambda. Inc. Without treatment. LSAB contain both native and denaturedclass I HLAs bound to the beads surfaces.

FIGS. 1A-1C show the reactivity of W6/32 and HC10 with LSAB. Monoclonalantibody W6/32 specifically binds to native class I HLA. Monoclonalantibody HC10 specifically binds to denatured class I HLA. FIGS. 1A-1Cdepict median fluorescence intensity (MFI) with respect to specificitiesfor A-locus (FIG. 1A), B-locus (FIG. 1B) and C-locus (FIG. 1C) class IHLA alleles. The level of MFI of the detection antibody corresponds tothe level of antibody binding to either native or denatured class I HLA.These figures show that LSAB are reactive to both W6/32 and HC10monoclonal antibodies.

To prepare the native class I HLA microbeads, denatured class I HLAs onthe LSAB were subjected to selective proteolytic digestion with trypsin,a serine protease. Trypsin was purchased in lyophilized form fromWorthington Biochemical Corporation of Lakewood, New Jersey (Cataloguecode TRL, Catalogue number LS00372).

The trypsin was reconstituted in 1× phosphate buffered saline (PBS),purchased from Irvine Scientific of Santa Ana. Calif., to workingconcentrations from about 0.00004% to about 0.04% weight/volume. Thenative class I HLA microbeads were exposed to Trypsin solution for about30 minutes at about 37 degrees C.

The trypsin solution was then neutralized using Trypsin NeutralizingSolution (TNS) purchased from Lonza Walkersville, Inc. of Walkersville,Md. (Catalogue number CC-5002). Stock TNS was added at about 2:1 byvolume after the about 30 minute digestion with trypsin. In otherinstances neutralization was accomplished by rapid dilution (about 1:2)using a mixture containing 1×PBS, about 0.1% bovine serum albumin (BSA).

Next, the microbeads were centrifuged at about 10,000 g for about twominutes and washed two times with about 1 mL of 1× LABScreen® washbuffer (Catalogue number LSPWABUF) The microbeads were subsequentlyincubated overnight in about 2% BSA at about 4° C. Then the microbeadswere washed three times with about 1 mL of LABScreen® wash buffer.

7.2 EXAMPLE 2 Assays of Native Class I HLA Microbeads

This example demonstrates the high purity of exemplary microbeadsprepared according to the methods described herein. To confirm thatproteolytic digestion removed denatured class I HLAs, a panel ofmicrobeads prepared according to Example 1 was incubated with eitherW6/32 or HC10 to detect native class I HLAs or denatured class I HLAsrespectively.

About 1 μl of antibody diluted in about 100 μl of about 1×PBS wereincubated with the native class I HLA microbeads for about 30 minutes atroom temperature on a shaker. The microbeads were then washed threetimes with about 1 mL of LABScreen® wash and then incubated with about100 μl of Phycoerythrin-conjugated (PE-conjugated) goat anti-mousesecondary antibody for about 30 minutes at room temperature on a shaker.Next, the microbeads were washed three times and analyzed by a Luminexflow machine.

FIGS. 2A-2C provide the reactivity, of W6/32 (specific for native classI HLA) and HC10 (specific for denatured class I HLA) with themicrobeads. The Y-axes represent the MFI and the X-axes representspecificities for A-locus (FIG. 2A). B-locus (FIG. 2B), and C-locus(FIG. 2C) class I HLA alleles. Raw data from the Luminex flow machinewas normalized to remove non-specific or background signal bysubtracting a sample negative control bead MFI from the MFI of allspecificities in the bead panel. All normalized values over 1,000 MFIwere considered positive. These figures show that the native class I HLAmicrobeads have low reactivity with HC10, indicating a low percentage ofdenatured class I HLAs remained on the native class I HLA microbeads.

7.3 Example 3 Purity of Native Class I HLA Microbeads

This example provides the purity of several exemplary samples of nativeclass I HLA microbeads prepared according to the methods describedherein. Tables 1-3 below show the percentage of denatured class I HLAremaining on microbeads made in accordance with the preparationprocedure provided in Example 1 above and assayed in accordance with theprocedure provided in Example 2 above. The percentage of denatured classI HLA was calculated according to the formula: [HC10 MFI/(HC10 MFI+W6/32MFI)]*100. Data was taken from 98 samples (5 batches) of microbeads.

Table 1 below is data from native class I HLA microbead samples forspecificities of A-locus class I HLA alleles. The data show the averagepercentage of denatured class I HLA of 0.81% with a standard deviationof 0.97%.

TABLE 1 Percentage of Denatured HLA on Native Class I HLA Microbeads(A-Locus) % denatured class I Sample ID HLA A*0101 0.14 A*0201 0.1A*0203 0.08 A*0206 0.1 A*0301 0.39 A*1101 0.19 A*1102 0.21 A*2301 0.15A*2402 0.19 A*2403 0.2 A*2501 2.52 A*2601 2.34 A*2901 0.17 A*2902 0.27A*3001 0.14 A*3002 0.17 A*3101 0.07 A*3201 0.16 A*3301 2.13 A*3303 1.29A*3401 0.81 A*3402 1.95 A*3601 0.17 A*4301 0.13 A*6601 1.97 A*6602 0.9A*6801 2.28 A*6802 2.42 A*6901 3.11 A*7401 0.17 A*8001 0.28

Table 2 below is data from microbeads samples for specificities ofB-locus class I HLA alleles. The data show the average percentage ofdenatured class I HLA of 3.53% with a standard deviation of 3.86° A.

TABLE 2 Percentage of Denatured HLA on Native Class I HLA Microbeads(B-Locus) % denatured class I % denatured class I Sample ID HLA SampleID HLA B*0702 1.02 B*4403 0.3 B*0801 12.81 B*4501 0.58 B*1302 1.13B*4601 2.96 B*1401 6.64 B*4701 1.62 B*1402 3.67 8*4801 4.14 B*1501 4.98B*4901 0.56 B*1502 4.34 B*5001 0.76 B*1503 0.68 B*5101 5.59 B*1510 2.4B*5102 7.05 B*1512 1.58 B*5201 1.62 B*1513 8.92 B*5301 10.37 B*1516 5.16B*5401 2.93 B*1801 1.41 B*5501 2.16 B*2705 0.54 B*5601 1.83 B*2708 0.75B*5701 6.69 B*3501 4.6 B*5703 0.16 B*3701 0.29 B*5801 18.45 B*3801 4.4B*5901 12.12 B*3901 2.1 B*6701 1.06 B*4001 0.91 B*7301 2.23 B*4002 0.6B*7801 4.23 B*4101 1.26 B*8101 2.4 B*4201 2.2 B*8201 1.62 B*4402 0.31B*1301 0.78 B*1511 10.45 B*4006 1.07

Table 3 below is data from microbeads samples for specificities ofC-locus class I HLA alleles. The data show the average percentage ofdenatured class I HLA of 2.60% with a standard deviation of 1.24%.

TABLE 3 Percentage of Denatured HLA on Native Class I HLA Microbeads(C-Locus) % denatured class I Sample ID HLA CW*0102 3.71 CW*0202 2.22CW*0302 3.71 CW*0303 1.43 CW*0304 2.52 CW*0401 2.01 CW*0501 1.56 CW*06021.88 CW*0702 5.42 CW*0801 1.28 CW*1203 1.24 CW*1402 1.67 CW*1502 4.52CW*1601 2.99 CW*1701 2.15 CW*1802 3.33

Overall results for Tables 1-3 show an average percentage of denaturedclass I HLA of 2.51% with a standard deviation of 3.10% on the fivebatches of native class I HLA microbeads. These results demonstrate thatnative class I HLA microbeads can be made with high purity according tothe present methods.

7.4 Example 4 Reactivity of Native Class I HLA Microbeads

A further investigation was performed comparing the reactivity profileof antibodies in nonalloimmunized male sera to LSAB beads and microbeadsprepared in accordance with Example 1. FIGS. 3A-3C show the reactivityof three nonalloimmunized male sera with LSAB and native class I HLAmicrobeads for A-locus (FIG. 3A), B-locus (FIG. 3B), and C-locus (FIG.3C) HLA alleles. In all three cases (A-locus, B-locus and C-locus), theresults show high positive reactivity of antibodies in the sera withLSAB microbeads but not native class I HLA microbeads. For antibodyreactivity to the A-locus alleles tested, there was an average of a98.3% reduction in MFI when using native class I HLA microbeads comparedto LSAB beads. For antibody reactivity to the B-locus alleles tested,there was an average of a 94.3% reduction in MFI when using native classI HLA microbeads compared to LSAB beads. For antibody reactivity to theC-locus alleles tested, there was an average of a 89.9% reduction in MFIwhen using native class I HLA microbeads compared to LSAB beads. Theseresults indicate that antibody binding is primarily due to denaturedclass I HLA and not due to native class I HLA. As described above,denatured class I HLA on LSAB beads can provide false positive signalsand result in the unnecessary exclusion of potential donors. Thus, theseresults show that native class I HLA microbeads can provide moreaccurate detection of class I HLAs.

All references cited herein are incorporated herein by reference intheir entireties and for all purposes, to the same extent as if eachindividual publication or patent or patent application were specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes. Although the invention has been described indetail with respect to various preferred embodiments it is not intendedto be limited thereto, but rather those skilled in the art willrecognize that variations and modifications may be made therein whichare within the spirit of the invention and the scope of the appendedclaims.

1. A composition comprising at least 90% native human leukocyte antigensand at most 10% denatured human leukocyte antigens.
 2. The compositionof claim 1, wherein the human leukocyte antigens are selected from thegroup consisting of class I human leukocyte antigens, class II humanleukocyte antigens and combinations thereof.
 3. The composition of claim2, wherein the human leukocyte antigens are class I human leukocyteantigens.
 4. The composition of claim 1, wherein a east 90% of saidhuman leukocyte antigens are of the same allele.
 5. The composition ofclaim 1, wherein said human leukocyte antigens are linked to a solidsubstrate.
 6. The composition of claim 5, wherein the solid substrate isselected from the group consisting of a plurality of beads, a pluralityof microbeads, a plurality of microparticles, a plurality ofmicrospheres, a well, a membrane, a polymer, a filter and a microarrayand combinations thereof.
 7. The composition of claim 5 wherein thesolid substrate is a plurality of microbeads.
 8. The composition ofclaim 5, wherein the solid substrate comprises a material selected fromthe group consisting of silica, gold, latex, polystyrene, polysulfone,hydrogel, polyvinyl chloride, glass, and combinations thereof.
 9. Thecomposition of claim 5, wherein the solid substrate comprises adetectable label.
 10. The composition of claim 9, wherein the detectablelabel is a fluorescent dye, a radioactive label, a magnetic label, a barcode, or combinations thereof.
 11. The composition of claim 5, whereinsaid human leukocyte antigens are covalently linked to the solidsubstrate.
 12. The composition of claim 5 comprising a plurality of saidsolid substrates, wherein at least 90% of the human leukocyte antigenslinked to a particular solid substrate of the plurality are of the sameallele and each solid substrate of the plurality is linked to adifferent human leukocyte antigen allele with respect to the other solidsubstrates of the plurality.
 13. The composition of claim 12, whereinthe native and denatured human leukocyte antigens are class I humanleukocyte antigens.
 14. The composition of claim 12, wherein theplurality of solid substrates comprises four or more solid substrates.15. The composition of claim 12, wherein the plurality of solidsubstrates comprises eight or more solid substrates.
 16. The compositionof claim 12, wherein the plurality of solid substrates comprises 16 ormore solid substrates.
 17. The composition of claim 12, wherein theplurality of solid substrates comprises 32 or more solid substrates. 18.A method of making a composition comprising at least 90% native humanleukocyte antigens and at most 10% leukocyte antigens, comprising thesteps of: a. contacting a first composition comprising native anddenatured human leukocyte antigens with a serine protease, lipase,esterase, or amidase under conditions wherein the serine protease,lipase, esterase or amidase digests the denatured human leukocyteantigens; and b. neutralizing the serine protease, lipase, esterase oramidase to yield a resulting composition comprising at least 90% nativehuman leukocyte antigens and at most 10% leukocyte antigens.
 19. Themethod of claim 18, wherein said human leukocyte antigens comprisesnative and denatured class I human leukocyte antigens.
 20. The method ofclaim 18, wherein said human leukocyte antigens are linked to one ormore solid substrates.
 21. The method of claim 20, wherein the humanleukocyte antigens are linked to a plurality of microbeads ormicroparticles.
 22. The method of claim 18, wherein said firstcomposition is contacted with a serine protease.
 23. The method of claim22, wherein said serine protease is selected from the group consistingof trypsin, chymotrypsin, elastase, subtilisin or combinations thereof.24. The method of claim 18, wherein said first composition is contactedwith a lipase.
 25. The method of claim 24, wherein the lipase is aphospholipase.
 26. The method of claim 18, wherein said firstcomposition is contacted with an esterase.
 27. The method of claim 26,wherein the esterase is an acetylcholine esterase.
 28. The method ofclaim 26, wherein the esterase is a thioesterase.
 29. The method ofclaim 18, wherein said first composition is contacted with an amidase.30. A method of screening for antibodies that bind native humanleukocyte antigens, comprising the steps of: a. contacting a sample withthe composition of claim 5; and b. detecting binding of an antibody tothe composition, wherein binding of an antibody to said composition isindicative of antibodies specific for said native human leukocyteantigens.
 31. The method of claim 30, wherein the detecting binding ofan antibody is performed using flow cytometry.
 32. The method of claim30, wherein the detecting binding of the antibody is performed using asecondary antibody.
 33. The method of claim 32, wherein the secondaryantibody comprises a label selected from the group consisting of aradioactive label, a fluorescent label, an enzymatic label, an avidinlabel a biotin label and combinations thereof.
 34. The method of claim30, wherein the composition comprises a plurality of microbeads ormicroparticles.
 35. The method of claim 34, wherein the compositioncomprises a plurality of microbeads, wherein each microbead of theplurality comprises a detectable label.
 36. The method of claim of claim35, wherein the detectable label is a fluorescent dye, a radioactivelabel, a magnetic label, or a bar code.
 37. A kit comprising thecomposition of claim 5 and a reagent for detecting the binding ofantibody to the composition.
 38. The kit of claim 37, wherein thereagent comprises a secondary antibody.
 39. The kit of claim 38, whereinthe secondary antibody comprises a label selected from the groupconsisting of a radioactive label, a fluorescent label, an enzymaticlabel, an avidin label and a biotin label.